The overall goal of this project is to develop a practical HIV immunogen that will give rise to long-lasting and broadly reactive anti-HIV T helper cell (Th) and MHC-restricted CD8+ cytotoxic T lymphocyte (CTL) responses. Recent studies have demonstrated that immunization with live vectors expressing whole HIV proteins or infection with live HIV gives rise to CD8+ CTL that recognize select immunodominant epitopes. A major hypothesis to be tested is that immunization with either HIV Th-CTL peptides or linear arrays of HIV Th-CTL epitopes expressed in modified vaccinia ankara (MVA) vaccinia will induce immunodominant CTL responses to those epitopes that are otherwise non-dominant in the context of whole proteins. Specific aims are: 1) to study multivalent Th-CTL HIV peptide immunogens comprised of Th and CTL determinants with tests for immunogenicity in mice, rhesus monkeys and humans; 2) to determine the immunogenicity of MVA that express the genes of the linear arrays of Th- CTL peptides in Aim 1, and compare the immunogenicity of each of the Th and CTL epitopes when expressed in MVA versus mixtures of Th-CTL synthetic peptides; 3) to induce durable protective cellular anti-HIV immune responses, we will augment the pool size of effector CTL (eCTL) and memory (precursor) CTL (pCTL) present after immunization with HIV immunogens by addition to adjuvant formulations, chemokines for T and dendritic cells, ligands for dendritic cell CD40, and angiogenic factors to augment immune cell recruitment into the immunization site. Specific aim 4 will test the optimal MVA/peptide immunogen prime/boast immunization protocol and optimal formulation and route of immunization in a Phase 1 human clinical trial. In aim 5, David Montefiori will perform neutralization assays on sera from animals immunized with env- containing vaccines from the first and second projects. This work should provide key information for understanding how to induce salutary anti- HIV T helper and CTL immune responses in humans.